Base sequence for protein expression and method for producing protein using same

ABSTRACT

To provide a base sequence for protein expression that can increase the yield of protein such as diastatic enzyme by further activating a promoter of a particular gene. A base sequence  1  for protein expression includes: a gene  3  encoding protein  2 ; a promoter  4  of the gene  3 , the promoter being linked upstream of the gene  3 ; and a cis element  5  whose activity is improved by an artificial transcription factor  6 , the cis element being linked further upstream of the promoter  4 . The cis element  5  is represented by SEQ ID NO: 1.

TECHNICAL FIELD

The present invention relates to a base sequence for protein expressionfor use in the production of protein such as diastatic enzyme using kojimold, and a method for producing protein using the same.

BACKGROUND ART

Heretofore, it has been known that a base sequence for proteinexpression in which a cis element consisting of a particular basesequence is linked to a promoter of a particular gene that yieldsprotein, when producing protein such as a diastatic enzyme using kojimold (see e.g., Patent Literatures 1 and 2). The conventional basesequence for protein expression can improve the activity of the promoterand can increase the yield of the protein, by linking the cis element tothe promoter.

For example, Patent Literature 1 describes a technique of using enhancerDNA consisting of a XlnR/Ace2 binding sequence and a Hap complex bindingsequence as a cis element and linking 12 cis elements upstream (on the5′-terminal side) of a promoter of tef1 gene. According to PatentLiterature 1, in this way, GUS activity by the promoter is reported tobe improved approximately 4.9 times under solid culture conditions withwheat bran as a carbon source.

Also, Patent Literature 2 describes a technique of using enhancer DNAlocated at a promoter of α-glucosidase gene of koji mold (Aspergillusoryzae) as a cis element and linking 12 such cis elements upstream (onthe 5′-terminal side) of the promoter. According to Patent Literature 2,in this way, GUS activity by the promoter is reported to be improvedapproximately 6 times under culture conditions with starch as a carbonsource.

CITATION LIST Patent Literature

[PTL 1]

Japanese Patent Application Laid-Open No. 2012-75369

[PTL 2]

Japanese Patent No. 3343567

SUMMARY OF INVENTION Technical Problem

However, the conventional base sequence for protein expression merelylinks a cis element consisting of a particular base sequence to apromoter of a particular gene and is thus desired to be furthermodified.

In light of these circumstances, an object of the present invention isto provide a base sequence for protein expression that can increase theyield of protein such as diastatic enzyme by further activating apromoter of a particular gene, and a method for producing protein usingthe same.

Solution to Problem

In order to attain the object, the base sequence for protein expressionof the present invention is a base sequence for protein expressioncomprising: a gene encoding protein; a promoter of the gene, thepromoter being linked upstream of the gene; and a cis element whoseactivity is improved by an artificial transcription factor, the ciselement being linked further upstream of the promoter, wherein the ciselement is represented by SEQ ID NO: 1, and wherein the artificialtranscription factor comprises a DNA binding domain comprising a basesequence of upstream 1 to 118 aa of a transcription factor KojR and anactive domain comprising a base sequence of downstream 150 to 604 aa ofa transcription factor AmyR, and the active domain is linked downstreamof the DNA binding domain, and is represented by SEQ ID NO: 2.

According to the base sequence for protein expression of the presentinvention, the activity of the cis element represented by SEQ ID NO: 1linked upstream of the promoter can be improved by the artificialtranscription factor represented by SEQ ID NO: 2, and the activity ofthe promoter can be further improved by the cis element whose activityhas been improved. As a result, the activity of the gene is improved bythe promoter whose activity has been improved, so that the yield of theprotein encoded by the gene can be increased.

The base sequence for protein expression of the present inventionpreferably comprises a base sequence for artificial transcription factorexpression comprising: a gene encoding the artificial transcriptionfactor represented by SEQ ID NO: 2; and a promoter of the gene, thepromoter being linked upstream of the gene. According to the basesequence for artificial transcription factor expression, the activity ofthe gene encoding the artificial transcription factor represented by SEQID NO: 2 is improved by the promoter of the gene so that the artificialtranscription factor encoded by the gene is produced.

For the base sequence for protein expression of the present invention,it is required that at least one cis element represented by SEQ ID NO: 1should be linked upstream of the promoter. Preferably, the cis elementis linked, for example, at any number in a range of 1 to 10, upstream ofthe promoter.

The expression vector of the present invention comprises the basesequence for protein expression of the present invention. According tothe expression vector of the present invention, a transformantcomprising the base sequence for protein expression of the presentinvention can be produced.

The transformant of the present invention comprises the base sequencefor protein expression of the present invention. According to thetransformant of the present invention, the yield of the protein encodedby the gene can be increased.

For the transformant of the present invention, it is preferred that kojimold should be used as a host cell, and it is more preferred that thekoji mold should be an Aspergillus oryzae HO2 strain (National Instituteof Technology and Evaluation, Patent Microorganisms Depositary, #122,2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Nov.12, 2013, Deposition No.: NITE BP-01750), or an Aspergillus oryzae HO4strain (National Institute of Technology and Evaluation, PatentMicroorganisms Depositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi,Chiba, Japan, Deposition Date: Dec. 9, 2014, Deposition No.: NITEBP-01980).

The method for producing a protein according to the present inventioncomprises culturing a transformant comprising the base sequence forprotein expression of the present invention, and recovering the proteinencoded by the gene overexpressed by the base sequence for proteinexpression, from the medium or the inside of the transformant after theculture.

The base sequence for protein expression of the present invention canincrease the yield of the protein encoded by the gene, as mentionedabove. Accordingly, when the transformant comprising the base sequencefor protein expression of the present invention is cultured, theproduced protein accumulates in the medium or the transformant after theculture. Therefore, the protein can be recovered.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an illustrative diagram schematically showing theconfiguration and effect of a base sequence for protein expression ofthe present invention.

FIG. 2 is an illustrative diagram schematically showing a predictedstructure of a transcription factor KojR.

FIG. 3 is an illustrative diagram schematically showing a predictedstructure of a transcription factor AmyR.

FIG. 4 is a graph showing a relative value of GUS activity when atransformant of the present invention was cultured for 60 hours withdextrin as a substrate.

FIG. 5 is a graph showing GUS activity when the transformant of thepresent invention was cultured for 90 hours with dextrin as thesubstrate.

FIG. 6 is a graph showing GUS activity when the transformant of thepresent invention was cultured for 60 hours with glucose as a substrate.

FIG. 7 is a graph showing CBH1 production amount when the transformantof the present invention was cultured for 6 days with dextrin as asubstrate

DESCRIPTION OF EMBODIMENTS

Next, the embodiments of the present invention will be described furtherspecifically with reference to the attached drawings.

As shown in FIG. 1, a base sequence 1 for protein expression of thepresent embodiment comprises: a protein gene 3 encoding a desiredprotein 2; a promoter 4 linked upstream (on the 5′-terminal side) of theprotein gene 3; and a cis element 5 linked upstream (on the 5′-terminalside) of the promoter 4.

The protein 2 is, for example, a diastatic enzyme. The protein gene 3may be any gene which encodes the protein 2.

The cis element 5 is composed of a base sequence comprising enhancer DNAlocated at a promoter of kojT gene, and the base sequence isgacggaaaagtcgggtagat (SEQ ID NO: 1). In the base sequence 1 for proteinexpression, 1 to 10, for example, 8 cis elements 5 are linked upstreamof the promoter 4.

The base sequence 1 for protein expression also comprises a basesequence 9 for artificial transcription factor expression comprising: anartificial transcription factor gene 7 encoding an artificialtranscription factor 6; and a promoter 8 linked upstream (on the5′-terminal side) of the artificial transcription factor gene 7. Theactivity of the cis element 5 is improved by the artificialtranscription factor 6.

The artificial transcription factor 6 is prepared from a transcriptionfactor KojR 11 shown in FIG. 2 and a transcription factor AmyR 21 shownin FIG. 3. The transcription factors KojR 11 and AmyR 21 aretranscription factors both classified into Cys6 cysteine-Zinc clustertype, among transcription factors having a zinc-coordinating DNA bindingdomain (Zn_Cluster).

As shown in FIG. 2, the transcription factor KojR 11 comprises upstream(5′-terminal side) Zn_Cluster 12 and comprises downstream (3′-terminalside) MHR 13 which is a highly homologous region common in transcriptionfactors classified in Cys6 cysteine-Zinc cluster type. In this context,the transcription factor KojR 11 is composed of a base sequence of 555aa in full length. The Zn_Cluster 12 is composed of a base sequence of15 to 45 aa. The MHR 13 is composed of a base sequence of 148 to 281 aa.

In the transcription factor KojR 11, a DNA binding domain associatedwith binding to the cis element 5 is predicted to reside in a region 14comprising the upstream Zn_Cluster 12. Examples of a candidate region ofthe DNA binding domain can include a region composed of a base sequenceof 1 to 118 aa, a region composed of a base sequence of 1 to 195 aa, anda region composed of a base sequence of 1 to 239 aa.

On the other hand, as shown in FIG. 3, the transcription factor AmyR 21comprises upstream (5′-terminal side) Zn_Cluster 22 and comprises adownstream (3′-terminal side) region 23 comprising an active domain. Inthis context, the transcription factor AmyR 21 is composed of a basesequence of 604 aa in full length. The Zn_Cluster 22 is composed of abase sequence of 13 to 52 aa.

Examples of a candidate region of the active domain in the transcriptionfactor AmyR 21 can include a region composed of a base sequence of 113to 604 aa, a region composed of a base sequence of 150 to 604 aa, aregion composed of base sequence of 219 to 604 aa, and a region composedof a base sequence of 257 to 604 aa.

Accordingly, the artificial transcription factor of the presentembodiment has a configuration (SEQ ID NO: 2) in which an active domaincomprising a base sequence of downstream 150 to 604 aa of thetranscription factor AmyR is linked downstream of a DNA binding domaincomprising a base sequence of upstream 1 to 118 aa of the transcriptionfactor KojR.

According to the base sequence 1 for protein expression of the presentembodiment, as shown in FIG. 1, the artificial transcription factor 6encoded by the artificial transcription factor gene 7 whose activity hasbeen improved by the promoter 8 in the base sequence 9 for artificialtranscription factor expression is produced, and the produced artificialtranscription factor 6 binds to the cis element 5. The activity of thecis element 5 is improved by the binding to the artificial transcriptionfactor 6. The activity of the promoter 4 is improved by the cis element5 whose activity has been improved.

Then, the activity of the protein gene 3 is improved by the promoter 4whose activity has been improved, so that the protein 2 encoded by theprotein gene 3 whose activity has been improved, is produced. As aresult, the base sequence 1 for protein expression of the presentembodiment can increase the yield of the protein 2.

Next, Examples of the present invention will be shown.

EXAMPLE 1

(Construction of Transformant Introduced with Artificial TranscriptionFactor Gene (1))

In this Example, first, the genomic DNA gene of an Aspergillus oryzaeHO2 strain (National Institute of Technology and Evaluation, PatentMicroorganisms Depositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi,Chiba, Japan, Deposition Date: Nov. 12, 2013, Deposition No.: NITEBP-01750) was used as a template in PCR to amplify an upstream sequenceof tppA gene using primers 1 and 2, its downstream sequence usingprimers 3 and 4, a tef1 promoter gene using primers 5 and 6, an agdAterminator gene using primers 7 and 8, and a gene fragment for markerrecycling using primers 9 and 10, while the genomic DNA gene of anAspergillus awamori HA1 strain (National Institute of Technology andEvaluation, Patent Microorganisms Depositary, #122, 2-5-8Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Nov. 12,2013, Deposition No.: NITE BP-01751) was used as a template in PCR toamplify a gene cassette for pyrG gene expression using primers 11 and12. DNA polymerase (manufactured by Toyobo Co., Ltd., product name: KODFX neo) was used in each PCR amplification. The amplification productswere each purified using a purification kit (manufactured by QiagenN.V., product name: QIAquick PCR purification kit) to obtain a total of6 gene fragments.

Next, an E. coli-derived plasmid pMD20 (manufactured by Takara Bio Inc.)was used as a template in PCR to amplify a gene fragment derived fromthe plasmid using primers 13 and 14 and the DNA polymerase. Theamplification product was purified using the purification kit to obtainthe gene fragment.

Next, these 7 gene fragments were sequentially treated with a cloningkit (manufactured by Takara Bio Inc., product name: In-Fusion HD Cloningkit) and used in the transformation of an E. coli HST08 strain(manufactured by Takara Bio Inc.) to construct a plasmid pPT.

Next, the plasmid pPT was treated with a restriction enzyme SmaI(manufactured by Takara Bio Inc.) at 30° C. and purified using thepurification kit to obtain the restriction treatment product of theplasmid (gene fragment).

Next, the genomic DNA gene of an Aspergillus oryzae HO2 strain (NationalInstitute of Technology and Evaluation, Patent MicroorganismsDepositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan,Deposition Date: Nov. 12, 2013, Deposition No.: NILE BP-01750) was usedas a template in PCR to amplify a DNA binding domain of a transcriptionfactor KojR using primers 15 and 16 and an active domain of atranscription factor AmyR using primers 17 and 18. The DNA polymerasewas used in each PCR amplification. The amplification products were eachpurified using the purification kit to obtain the DNA binding domain andthe active domain.

Next, the DNA binding domain and the active domain were treated with thecloning kit and used in the transformation of an E. coli HST08 strain toconstruct a plasmid carrying an artificial transcription factor gene inwhich the DNA binding domain and the active domain were joined together.

The plasmid carrying the artificial transcription factor gene was usedas a template in PCR to amplify a gene fragment for koji moldtransformation using primers 19 and 20 using DNA polymerase(manufactured by Toyobo Co., Ltd., product name: KOD-plus-neo). Theamplification product was purified using the purification kit to obtainthe gene fragment for koji mold transformation.

Next, an Aspergillus oryzae HO2 strain (National Institute of Technologyand Evaluation, Patent Microorganisms Depositary, #122, 2-5-8Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Nov. 12,2013, Deposition No.: NITE BP-01750) was transformed with the genefragment for koji mold transformation according to the standard methodof the PEG-calcium technique. Subsequently, the obtained transformantswere screened for a strain capable of growing in a CD plate medium toobtain a transcription factor-producing strain.

Next, the transcription factor-producing strain was inoculated at 1×10⁶cells/plate to a CD medium supplemented with fluoroorotic acidmonohydrate (manufactured by Wako Pure Chemical Industries, Ltd.) (finalconcentration: 1 mg/mL) and uridine (manufactured by Sigma-Aldrich Inc.)(final concentration: 20 mM) and screened for a strain capable ofgrowing therein to obtain a uridine-auxotrophic transcriptionfactor-producing strain.

The base sequences of the primers 1 to 20 are shown in Table 1.

TABLE 1 SEQ Primer ID No. Base sequence 5′→3′ NO Remarks  1ccggctcgtatgttgctggaccaaccgccaaggttag  3 Upstream sequence of tppA gene 2 actgaattgcaattaatggcggacaatg  4 Upstream sequence of tppA gene  3tgtctcggaccttacgtgtcttagatgcgactcaatacaactgttc  5 Downstream sequenceof tppA gene  4 tgggtaacgccagggttgaggctgaagacttaaatacgacattgc  6Downstream sequence of tppA gene  5ctgttacgcttccccgggtttgaaggtggtgcgaactttgtagttc  7 tef1 promoter gene  6gtaaggtccgagacagtaagggattgatc  8 tef1 promoter gene  7taattgcaattcagtagtaacccattcccggttctctagctg  9 agdA terminator gene  8gtaacgccagggcccggggaagcgtaacaggatagcctagacccac 10 agdA terminator gene 9 ctgcaggatgattagcgtgcaaaccaagcaaacaagcatc 11 Gene fragment formarker recycling 10 actgaattgcaattaatggcggacaatg 12 Gene fragment formarker recycling 11 taattgcaattcagtgcaagctcgagcatccaactaaactag 13Gene cassette for prG gene expression 12tgggtaacgccagggcccgggctaatcatcctgcagctccgtcattg 14 Gene cassette for prGgene expression 13 ccctggcgttacccaacttaatcg 15 Plasmid-derived genefragment 14 caacatacgagccggaagcataaagtg 16 Plasmid-derived gene fragment15 cgcaccaccttcaaaatgtcgttgaataccgacgattccggtc 17 DBD of transcriptionfactor kojR 16 acctaggttccagctaaacccgtacac 18 DBD of transcriptionfactor kojR 17 atcctgttacgcttctcaaaacgaaatctcctccccagcc 19AD of transcription factor AmyR 18 agctggaacctaggtgcactccccgcgccac 20AD of transcription factor AmyR 19 cagtgagcgcaacgcaattaatgtgagttag 21Gene fragment for koji mold transformation 20gggatgtgctgcaaggcgattaagttg 22 Gene fragment for kojimold transformation[Construction of GUS-producing Strain with Cis Elements Linked]

First, a first gene fragment in which: 4 cis elements of SEQ ID NO: 1were linked in tandem; restriction enzyme sites SphI and BamHI wereadded on the 5′-terminal side thereof; and BglII and NcoI sites wereadded on the 3′-terminal side thereof was prepared by oligo synthesis.

Next, the first gene fragment and a plasmid pPEA2 containing anAspergillus oryzae-derived enoA promoter were each fragmented bytreatment with restriction enzymes SphI and NcoI. These fragments weresubjected to ligation reaction, and E. coli was then transformed withthe ligation product to construct a plasmid pEA4K.

Next, the gene fragment was treated with a restriction enzyme BamHI,while the plasmid pEA4K was treated with restriction enzymes BglII andNcoI. These two treatment products were subjected to ligation reaction,and E. coli was then transformed with the ligation product to constructa plasmid pEA8K.

Next, the plasmid pEA8K was used as a template in PCR amplificationusing primers 21 and 22 and DNA polymerase (manufactured by Toyobo Co.,Ltd., product name: KOD-plus-). The amplification product was purifiedusing a purification kit (manufactured by Promega Corp., product name:Wizard SV Gel and PCR Clean-Up System) to obtain a second gene fragment.

Next, the genomic DNA of Aspergillus oryzae was used as a template inPCR amplification using primers 23 and 24 and DNA polymerase(manufactured by Toyobo Co., Ltd., product name: KOD-plus-). Theamplification product was purified using a purification kit(manufactured by Promega Corp., product name: Wizard SV Gel and PCRClean-Up System) to obtain a third gene fragment.

Next, the second gene fragment and the third gene fragment were used asa template in fusion PCR using primers 22 and 24 to prepare a fourthgene fragment in which the second gene fragment and the third genefragment were joined together.

Next, a restriction enzyme-treated plasmid pPPG introduced with an E.coli-derived plasmid pMD20 (manufactured by Takara Bio Inc.) carryingupstream 1000 bp of Aspergillus oryzae-derived pyrG gene, an Aspergillusoryzae-derived pyrG expression cassette, and an E. coli-derived uidAgene was subjected to ligation reaction with a gene fragment for markerrecycling obtained by PCR-amplifying a plasmid pPPG as a template usingprimers 25 and 26 and DNA polymerase (manufactured by Toyobo Co., Ltd.,product name: KOD-plus-) and purifying the amplification product using apurification kit (manufactured by Promega Corp., product name: Wizard SVGel and PCR Clean-Up System). Then, E. coli was transformed with theligation product to construct a plasmid pPPRG.

Next, the plasmid pPPRG was used as a template in PCR amplificationusing primers 27 and 28 and DNA polymerase (manufactured by Toyobo Co.,Ltd., product name: KOD-plus-). The amplification product was purifiedusing a purification kit (manufactured by Promega Corp., product name:Wizard SV Gel and PCR Clean-Up System) to obtain a fifth gene fragment.

The fourth gene fragment and the fifth gene fragment were used as atemplate in fusion PCR using primers 24 and 27 to prepare a ciselement-linked GUS (β-glucuronidase) production cassette gene fragmentin which the fourth gene fragment and the fifth gene fragment werejoined together.

Next, the uridine-auxotrophic transcription factor-producing strain wastransformed using the cis element-linked GUS production cassette genefragment according to the standard method of the PEG-calcium technique.Subsequently, the obtained transformants were screened for a straincapable of growing in a CD plate medium to obtain a GUS-producing strainwith 8 cis elements linked in tandem.

The base sequences of the primers 21 to 28 are shown in Table 2.

TABLE 2 Primer SEQ ID No. Base sequence 5′→3′ NO 21ccgctgctaggcgcgccgtgcactatagggcgaattgggc 23 22tggggtttctacaggacgtaacattttgacgagctgcggaatt 24 23cacggcgcgcctagcagcgggtagtggtggatacgtactcctt 25 24ttcaggtcacgttctaagcttatcag 26 25 cccccctccggatgatgtagaagttgctcggtagctg27 26 cccccctccggacaattgccgcgaaaaattaaattg 28 27ccagaggtgactttatccaagatt 29 28caattccgcagctcgtcaaaatgttacgtcctgtagaaacccca 30[GUS Activity Measurement Method]

The GUS-producing strain with 8 cis elements linked in tandem wascultured in a CD plate medium for 1 week to form spores. The spores wererecovered using 0.01% POLYSORBATE 20 (manufactured by Wako Pure ChemicalIndustries, Ltd.) to obtain a spore suspension.

Next, 50 mL of a PD medium (2 mass/volume % of dextrin, 1 mass/volume %of polypeptone, 0.1 mass/volume % of casamino acid, 0.5 mass/volume % ofpotassium dihydrogen phosphate, 0.05 mass/volume % of magnesium sulfate,and 0.1 mass/volume % of sodium nitrate) was placed in a 200 mLErlenmeyer flask, to which the spores were then inoculated at a finalspore concentration of 1×10⁵/mL.

Next, liquid culture was performed at 30° C. for 60 hours. After thecompletion of the culture, the bacterial cells were disrupted, and thedisrupted powder was suspended in a buffer for intracellular proteinextraction having the composition given below to obtain an extract.

[Composition of Buffer for Intracellular Protein Extraction]

NaH₂PO₄.2H₂O (MW=156.01) (pH 7) 1.56 g (50 mM)

0.5 M EDTA 4 mL (10 mM)

Nonionic surfactant (manufactured by Sigma-Aldrich Inc., product name:Triton X-100) 0.2 g (0.1%)

N-Laurylsarcosinate Na 0.2 g (0.1%)

β-mercaptoethanol (MW=78.13) 142 μL (10 mM)

Distilled water 200 mL

Next, the extract was added to a buffer for GUS activity measurementhaving the composition given below and reacted at 37° C. for 15 minutes.Then, the absorbance was measured at a wavelength of 415 nm to calculatean activity value (U). 1 U means the amount of the enzyme necessary forforming 1 mM PNP from PNP-Glucuronide (purine nucleosidephosphorylase-glucuronic acid inclusion) at 37° C. for 1 minute.

[Composition of Buffer for GUS Activity Measurement]

NaH₂PO₄.2H₂O (MW=156.01) (pH 7) 1.56 g (50 mM)

β-mercaptoethanol (MW=78.13) 142 μL (10 mM)

Nonionic surfactant (manufactured by Sigma-Aldrich Inc., product name:Triton X-100) 0.2 g (0.1%)

p-Nitrophenyl β-D-glucuronic acid inclusion (MW=315.23) 63 mg (1 mM)

Distilled water 200 mL

Next, the amount of the protein contained in the extract was measuredusing protein assay CBB solution (manufactured by Nacalai Tesque, Inc.),and the activity value was divided by the amount of the protein tocalculate GUS activity (U/mg). The results are shown as a relative valueof GUS activity in FIG. 4.

Also, GUS activity (U/mg) when the liquid culture was performed at 30°C. for 90 hours is shown in FIG. 5.

COMPARATIVE EXAMPLE 1

In this Comparative Example, a GUS-producing strain was constructed intotally the same way as in Example 1 except that the artificialtranscription factor gene was not introduced and no cis element waslinked.

Next, GUS activity was measured in totally the same way as in Example 1except that the GUS-producing strain obtained in this ComparativeExample was used.

A relative value of GUS activity (U/mg) when the liquid culture wasperformed at 30° C. for 60 hours is shown in FIG. 4. GUS activity (U/mg)when the liquid culture was performed at 30° C. for 90 hours is shown inFIG. 5.

From FIG. 4, when the GUS activity (U/mg) in which the liquid culturewas performed at 30° C. for 60 hours with dextrin as a substrate isdefined as 1 for the GUS-producing strain of Comparative Example 1 inwhich the artificial transcription factor gene was not introduced and nocis element was linked, it is obvious that 25.1 times GUS activity canbe obtained in the GUS-producing strain of Example 1.

From FIG. 5, when the GUS activity (U/mg) when the liquid culture wasperformed at 30° C. for 90 hours with dextrin as a substrate is definedas 1 for the GUS-producing strain of Comparative Example 1 in which theartificial transcription factor gene was not introduced and no ciselement was linked, it is obvious that 28 times GUS activity can beobtained in the GUS-producing strain of Example 1.

EXAMPLE 2

In this Example, GUS activity (U/mg) was calculated in totally the sameway as in Example 1 except that 50 mL of a PG medium (2 mass/volume % ofglucose, 1 mass/volume % of polypeptone, 0.1 mass/volume % of casaminoacid, 0.5 mass/volume % of potassium dihydrogen phosphate, 0.05mass/volume % of magnesium sulfate, and 0.1 mass/volume % of sodiumnitrate) was placed in a 200 mL Erlenmeyer flask, to which the spores ofthe GUS-producing strain harboring 8 cis elements linked in tandemobtained in Example 1 were then inoculated at a final sporeconcentration of 1×10⁵/mL, followed by liquid culture at 30° C. for 60hours. The results are shown in FIG. 6.

COMPARATIVE EXAMPLE 2

In this Comparative Example, GUS activity was measured in totally thesame way as in Example 2 except that the GUS-producing strain obtainedin Comparative Example 1 was used. GUS activity (U/mg) when the liquidculture was performed at 30° C. for 60 hours is shown in FIG. 6.

From FIG. 6, when the GUS activity (U/mg) in which the liquid culturewas performed at 30° C. for 60 hours with glucose as a substrate isdefined as 1 for the GUS-producing strain of Comparative Example 2 inwhich the artificial transcription factor gene was not introduced and nocis element was linked, it is obvious that 14.8 times GUS activity canbe obtained in the GUS-producing strain of Example 2.

EXAMPLE 3

(Construction of Transformant Introduced with Artificial TranscriptionFactor Gene (2))

In this Example, first, the genomic DNA gene of an Aspergillus oryzaeHO4 strain (National Institute of Technology and Evaluation, PatentMicroorganisms Depositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi,Chiba, Japan, Deposition Date: Dec. 9, 2014, Deposition No.: NITEBP-01980) was used as a template in PCR to amplify an upstream sequenceof ligD gene using primers 29 and 30, its downstream sequence usingprimers 31 and 32, a marker recycling sequence using primers 33 and 34,and a pyrG gene using primers 35 and 36. DNA polymerase (manufactured byToyobo Co., Ltd., product name: KOD FX neo) was used in each PCRamplification. The amplification products were each purified using apurification kit (manufactured by Qiagen N.V., product name: QIAquickPCR purification kit) to obtain a total of 4 gene fragments.

Next, an E. coli-derived plasmid pMD20 (manufactured by Takara Bio Inc.)was used as a template to obtain a gene fragment derived from theplasmid using primers 13 and 14.

Next, gene fragment of upstream sequence of ligD gene, gene fragment ofits downstream sequence, and gene fragment of plasmid pMD20 were treatedwith a cloning kit (manufactured by Takara Bio Inc., product name:In-Fusion HD Cloning kit) and used in the transformation of an E. coliHST08 strain (manufactured by Takara Bio Inc.) to construct a plasmidpM-Ao Δ ligD.

Next, the plasmid pM-Ao Δ ligD was treated with the restriction enzymeSmaI (manufactured by Takara Bio Inc.) at 30° C. and purified using thepurification kit to obtain the restriction treatment product of theplasmid pM-Ao Δ ligD (gene fragment).

Next, gene fragment of plasmid pM-Ao Δ ligD and gene fragment of pyrGgene were treated with the cloning kit and used in the transformation ofan E. coli HST08 strain (manufactured by Takara Bio Inc.) to construct aplasmid pM-Ao Δ ligD::pyrG in which pyrG gene was introduced between theupstream sequence of ligD gene and its downstream sequence.

Next, the plasmid pM-Ao Δ ligD::pyrG was treated with the restrictionenzyme SmaI (manufactured by Takara Bio Inc.) at 30° C. and purifiedusing the purification kit to obtain the restriction treatment productof the plasmid pM-Ao Δ ligD::pyrG (gene fragment).

Next, gene fragment of plasmid pM-Ao Δ ligD::pyrG and gene fragment ofthe marker recycling sequence were treated with the cloning kit and usedin the transformation of an E. coli HST08 strain (manufactured by TakaraBio Inc.) to construct a plasmid pM-Ao Δ ligD::pyrGR in which the markerrecycling sequence was introduced between the downstream sequence ofligD gene and the pyrG gene.

Next, the plasmid pM-Ao Δ ligD::pyrGR was treated with the restrictionenzyme SmaI (manufactured by Takara Bio Inc.) at 30° C. and purifiedusing the purification kit to obtain the restriction treatment productof the plasmid pM-Ao Δ ligD::pyrGR (gene fragment).

Next, the genomic DNA gene of an Aspergillus oryzae HO4 strain (NationalInstitute of Technology and Evaluation, Patent MicroorganismsDepositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan,Deposition Date: Dec. 9, 2014, Deposition No.: NITE BP-01980) was usedas a template in PCR to amplify an enoA promoter gene using primers 37and 38, and a plasmid introduced with the artificial transcriptionfactor gene in which the DNA binding domain and the active domain werejoined together prepared in Example 1 was used as a template in PCR toamplify an agdA terminator gene and an artificial transcription geneusing primers 39 and 40. The DNA polymerase was used in each PCRamplification. The amplification products were each purified using thepurification kit to obtain the DNA binding domain and the active domain.

Next, gene fragment of plasmid pM-Ao Δ ligD::pyrGR, gene fragment ofenoA promoter gene, agdA terminator gene, and artificial transcriptiongene were treated with the cloning kit and used in the transformation ofan E. coli HST08 strain (manufactured by Takara Bio Inc.) to construct aplasmid pM-Ao Δ ligD::pyrGR-TF1 in which artificial transcription factorgene was introduced between the upstream sequence of ligD gene and itsdownstream sequence.

The plasmidpM-Ao Δ ligD::pyrGR-TF1 carrying the artificial transcriptionfactor gene was used as a template in PCR to amplify a gene fragment forkoji mold transformation using primers 19 and 20 using the DNApolymerase. The amplification product was purified using thepurification kit to obtain the gene fragment for koji moldtransformation.

Next, an Aspergillus oryzae HO4 strain (National Institute of Technologyand Evaluation, Patent Microorganisms Depositary, #122, 2-5-8Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Dec. 9,2014, Deposition No.: NITE BP-01980) was transformed with the genefragment for koji mold transformation according to the standard methodof the PEG-calcium technique. Subsequently, the obtained transformantswere screened for a strain capable of growing in a CD plate medium toobtain a transcription factor-producing strain.

Next, the transcription factor-producing strain was inoculated at 1×10⁶cells/plate to a CD medium supplemented with fluoroorotic acidmonohydrate (manufactured by Wako Pure Chemical Industries, Ltd.) (finalconcentration: 1 mg/mL) and uridine (manufactured by Sigma-Aldrich Inc.)(final concentration: 20 mM) and screened for a strain capable ofgrowing therein to obtain a uridine-auxotrophic transcriptionfactor-producing strain.

The base sequences of the primers 29 to 40 are shown in Table 3.

TABLE 3 SEQ Primer ID No. Base sequence 5′→3′ NO Remarks 29tcgagctcgg tacccggtta ctgctctccc ttgatgatg 31 Upstream sequence ofligD gene 30 taggtagtga acctatttcg agagcag 32 Upstream sequence ofligD gene 31 taggttcact acctagcggc cgcacaggca ccttgcatca tcatc 33Downstream sequence of ligD gene 32ctctagagga tccccggacc gacgattcgt tgaagag 34 Downstreamsequence of ligD gene 33 aggtatcgaa ttcccgacga gctcgtacag atctttg 35marker recycling sequence 34ccatgggaaa tgcccgggag agcagagtgc atggaatact ag 36 marker recyclingsequence 35 gcactctgct ctcccggtgg tgggaaatct tgtatataat tgtgattg 37pyG gene 36 ccatgggaaa tgcccgggcg acactggaag aactgcttga agag 38 pyG gene37 cctgccgcga gatctgggca tttcccatgg gcctaaccca aatc 39enoA promoter gene 38 ccatgggaaa tgcccagatc tcgcggcagg gttgacacag ttgac40 enoA promoter gene 39 gcactctgct ctcccagtaa cccattcccg gttctctagc 41*1 40 atgtcgttga ataccgacga ttccggtc 42 *1 *1: agdA terminator gene,artificial transcription gene[Construction of CBH1 Producing Strain with Cis Elements Linked]

First, the genomic DNA gene of an Aspergillus oryzae HO4 strain(National Institute of Technology and Evaluation, Patent MicroorganismsDepositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan,Deposition Date: Dec. 9, 2014, Deposition No.: NITE BP-01980) was usedas a template in PCR to amplify an upstream sequence of pyrG gene usingprimers 41 and 42, its downstream sequence using primers 43 and 44. Acis element-linked GUS (β-glucuronidase) production cassette genefragment obtained in Example 1 was used as a template in PCR to amplifya cis element linked promoter gene using primers 45 and 46, agdAterminator gene using primers 47 and 48. The genomic DNA gene of anAcremonium cellulolyticus H1 strain (National Institute of Technologyand Evaluation, Patent Microorganisms Depositary, #122, 2-5-8Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Sep. 5,2011, Deposition No.: FERM BP-11508) was used as a template in PCR toamplify a cellobiohydrolase (cbh1) gene using primers 49 and 50. Thegenomic DNA gene of an Aspergillus awamori HA1 strain (NationalInstitute of Technology and Evaluation, Patent MicroorganismsDepositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan,Deposition Date: Nov. 12, 2013, Deposition No.: NITE BP-01751) was usedas a template in PCR to amplify a gene cassette for pyrG gene expressionusing primers 51 and 52. DNA polymerase was used in each PCRamplification. The amplification products were each purified using thepurification kit to obtain a total of 6 gene fragments.

Next, plasmid pMD20 (manufactured by Takara Bio Inc.) was treated with arestriction enzyme SmaI (manufactured by Takara Bio Inc.) at 30° C. andpurified using the purification kit to obtain the restriction treatmentproduct of the plasmid pMD20 (gene fragment).

Next, gene fragment of the upstream sequence of pyrG gene, gene fragmentof its downstream sequence, gene fragment of the cis element linkedpromoter gene, gene fragment of the agdA terminator gene, gene fragmentof the bch1 gene, gene fragment of the gene cassette for pyrG geneexpression, and gene fragment of the plasmid pMD20 were sequentiallytreated with the cloning kit and used in the transformation of an E.coli HST08 strain (manufactured by Takara Bio Inc.) to construct aplasmid pPPeA8-CBH1.

Next, the plasmid pPPeA8-CBH1 was used as a template in PCRamplification using primers 19 and 20 and using DNA polymerase. Theamplification product was purified using the purification kit to obtaina gene fragment for koji mold transformation (pyrG-CBH1 fragment).

Next, the Aspergillus oryzae HO4 strain (National Institute ofTechnology and Evaluation, Patent Microorganisms Depositary, #122, 2-5-8Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Dec. 9,2014, Deposition No.: NITE BP-01980) was transformed using the genefragment for koji mold transformation (pyrG-CBH1 fragment) according tothe standard method of the PEG-calcium technique. Subsequently, theobtained transformants were screened for a strain capable of growing ina CD plate medium to obtain a transformant which corresponds to thetransformant of Example 1.

The transformant is introduced with cellobiohydrolase (cbh1) gene in thechromosome, and is capable of producing cellobiohydrolase. Hereinafter,a transformant introduced with cbh1 gene in the chromosome and capableof producing cellobiohydrolase is abbreviated as “CBH1 producingstrain”.

The base sequences of the primers 41 to 52 are shown in Table 4.

TABLE 4 SEQ Primer ID No. Base sequence 5′→3′ NO Remarks 41ggatatcgga tccccccaga ggtgacttta tccaagattc cttc 43 Upstream sequence ofpyrG gene 42 caattgccgc gaaaaattaa attgaatcta tg 44 Upstream sequence ofpyrG gene 43 gtagtggtggatacgtactccttttatg 45 Downstream sequenceof pyrG gene 44 tcgagctcgg tacccttcag gtcacgttct aagcttatca gctg 46Downstream sequence of pyrG gene 45cgtatccaccactaccactatagggcgaattgggcccgac 47 PeA8 promotor gene 46gttcaaggcagacattttgacgagctgcggaattggtcag 48 PeA8 promotor gene 47ccaccactac cccgggaagc gtaacaggat agcctagacc 49 agdA terminator gene 48ctgcaggatg attagagtaa cccattcccg gttctctagc tg 50 agdA terminator gene49 atgtctgcct tgaactcttt caatatgtac aag 51 cbh1 gene 50atcctgttac gcttcctaca aacattgaga gtagtaaggg ttcacg 52 cbh1 gene 51ctaatcatcctgcagctccgtcattg 53 Cassette for pyrG gene expression 52ttttcgcggcaattggcaagctcgagcatccaactaaactag 54 Cassette for pyrG geneexpression[Enzyme Production Amount Measurement Method]

In order to measure the enzyme (cellobiohydrolase) production amount bythe CBH1 producing strain, first, the CBH1 producing strain with 8 ciselements linked in tandem was cultured in a CD plate medium for 1 weekto form spores. The spores were recovered using 0.01% POLYSORBATE 20(manufactured by Wako Pure Chemical Industries, Ltd.) to obtain a sporesuspension.

Next, 30 mL of a PD medium (2 mass/volume % of dextrin, 1 mass/volume %of polypeptone, 0.1 mass/volume % of casamino acid, 0.5 mass/volume % ofpotassium dihydrogen phosphate, 0.05 mass/volume % of magnesium sulfate,and 0.1 mass/volume % of sodium nitrate) was placed in a 100 mLErlenmeyer flask, to which the spores were inoculated at a final sporeconcentration of 1×10⁴/mL.

Next, liquid culture was performed at 30° C. for 6 days, to obtain aculture solution of CBH1 producing strain in which cellobiohydrolase(CBH1) was secreted and expressed in the culture medium.

Next, the CBH1 concentration in the culture solution was measured bySDS-PAGE analysis. BSA of 0.25 μg, 0.5 μg, and 2 μg were migrated at thesame time as the reference of the protein, and the CBH1 concentration inthe culture solution 10 μL was calculated by image analysis using animage automatic detection system (manufactured by BIO-RAD Corporation,product name: ChemiDoc XRS+system). The result is shown in FIG. 7.

COMPARATIVE EXAMPLE 3

In this Comparative Example, a CBH1 producing strain was constructed intotally the same way as in Example 3 except that the artificialtranscription factor gene was not introduced and no cis element waslinked.

Next, the CBH1 concentration in the culture solution was measured intotally the same way as in Example 3 except that the CBH1 producingstrain obtained in this Comparative Example was used. The result isshown in FIG. 7.

From FIG. 7, when the liquid culture was performed at 30° C. for 6 dayswith dextrin as a substrate, according to the CBH1 producing strain ofExample 3, it is obvious that 24.3 times enzyme (cellobiohydrolase,CBH1) can be produced compared to the CBH1 producing strain ofComparative Example 3 in which the artificial transcription factor genewas not introduced and no cis element was linked.

REFERENCE SIGNS LIST

-   1 base sequence for protein expression-   2 protein-   3 gene-   4 promoter-   5 cis element-   6 artificial transcription factor

Sequence Listing <110> HONDA MOTOR CO., LTD. <120>Base sequence for protein expression and production of protein <130>PCT160077 <160> 30 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA<213> Aspergillus oryzae <400> 1 gacggaaaag tcgggtagat 20 <210> 2 <211>1872 <212> DNA <213> Aspergillus oryzae <400> 2atgtcgttga ataccgacga ttccggtcgg ataaggaccc ggcaacgcgc caaaagagcg   60tgcgaaacgt gcaaactgcg caagaggaaa tgtgacggcc atgagccctg cacttactgc  120ttgcgatacg aatatcagtg cactttcaag cctcatccac ggagaaagcc tgcagcttcc  180aaatcttccg cacggcccag cgaggaagaa gactcaccaa agtttctcga cagagttgat  240gctaaccaag aacacatgga ggccaactca ggcaccgctt tcccccatct cctagggatg  300aggttgaacc cgcagggtgc tcccaaggtg tacgggttta gctggaacct aggtgcactc  360cccgcgccac gccgtctgtc gacgccaaac cttctagccc atgtcaatgt cttcctcaag  420tacctgttcc cgatcatgcc cgtcgtgaga caggaccagc tgcagcagga ctgccaccag  480ccggagcgct tgtctcccca acgctacgct ttcattgccg ctctatgcgc ggccacgcac  540atccaactga agctggacgg tgcagcaccg ggtcccgagg cggcttccgc gcgagccagc  600ctcgacggac atcctatgtt gtcgggagaa gaactcctgg ctgaagccgt gcgcgcaaga  660aaggaataca acgtggtcga cgaaattaac atggaaaacc tcctaacctc cttctttctc  720ttcgccgcct acggaaacct agacagacag gatcaggcct ggttctacct atgtcagacc  780acgtccatgg tcttcacact aggcctacaa cgggaatcca catactcgaa actaagcgtc  840gaggaagcag aagagaaaag gagagtattc tggctcttat tcgtcacaga aaggtaagaa  900aagaaaaaac tctactttcc caatcaccac cacgtaccaa aaataacacg aaaaaccaga  960ggctacgcat tacaacaagc aaaaccagtc atgctccgca actccatcca caaaccacag 1020gtcctgtgct cagacgaccc aatcctagcc tacggcttca tcaacctcat caacgtcttc 1080gaaaagctca gcccaaatct ctacgactgg gtctccgccg gcggcagcag cgcagacggc 1140gaccccccgc ctacttcttc tatccaatcc agtctcgcca agcaaatctc cctcgagggc 1200gtctccgaga tccagaaagt agacatcctc atcactcagc aatggctaca aaccatgatg 1260tggaaactct ccatgaccca cgtcacacag cccggctctc gcgatgacgc cgttctcccc 1320ttccacctgc ccgtgctagt cggcaaggcc gtcatgggcg tcatcgccgc ggcatcccaa 1380ggtgctgttg acgctcatgg tatcggaatg gtaagaaagc gaccttacct catcacaccc 1440tccctcatca gtcactcccc atcatctata cccgcaatct aacaaaaacc gcaggaacaa 1500aaactctacg acctcggcac ctccgtagcc gacgtctccc gctccctaag cacaaaagcc 1560gcccaccacc tcgccgaatc gaccatcgac ccccgagaac tcctctgggg cattctcaca 1620accctatccc gaatccgcgg ttcccaatca tacctcttcc cagcgctcgt cgagcaaagt 1680cgaggcatca tcagtttcga ctgttcgctt tccatcagtg actttctgcc ttcgtttggt 1740gggccgccgg ctattatgtg gcggacgggt gaatctgggt ttgatttatt ggggatcgcg 1800gatgatttgc aagagaggga gaatgagggt ggggagggga ttgtggtggc tggggaggag 1860atttcgatt ga 1872 <210> 3 <211> 37 <212> DNA <213> Aspergillus oryzae<400> 3 ccggctcgta tgttgctgga ccaaccgcca aggttag 37 <210> 4 <211> 28<212> DNA <213> Aspergillus oryzae <400> 4actgaattgc aattaatggc ggacaatg 28 <210> 5 <211> 46 <212> DNA <213>Aspergillus oryzae <400> 5tgtctcggac cttacgtgtc ttagatgcga ctcaatacaa ctgttc 46 <210> 6 <211> 45<212> DNA <213> Aspergillus oryzae <400> 6tgggtaacgc cagggttgag gctgaagact taaatacgac attgc 45 <210> 7 <211> 46<212> DNA <213> Aspergillus oryzae <400> 7ctgttacgct tccccgggtt tgaaggtggt gcgaactttg tagttc 46 <210> 8 <211> 29<212> DNA <213> Aspergillus oryzae <400> 8gtaaggtccg agacagtaag ggattgatc 29 <210> 9 <211> 42 <212> DNA <213>Aspergillus oryzae <400> 9taattgcaat tcagtagtaa cccattcccg gttctctagc tg 42 <210> 10 <211> 46<212> DNA <213> Aspergillus oryzae <400> 10gtaacgccag ggcccgggga agcgtaacag gatagcctag acccac 46 <210> 11 <211> 40<212> DNA <213> Aspergillus oryzae <400> 11ctgcaggatg attagcgtgc aaaccaagca aacaagcatc 40 <210> 12 <211> 28 <212>DNA <213> Aspergillus oryzae <400> 12 actgaattgc aattaatggc ggacaatg 28<210> 13 <211> 42 <212> DNA <213> Aspergillus awamorii <400> 13taattgcaat tcagtgcaag ctcgagcatc caactaaact ag 42 <210> 14 <211> 47<212> DNA <213> Aspergillus awamorii <400> 14tgggtaacgc cagggcccgg gctaatcatc ctgcagctcc gtcattg 47 <210> 15 <211> 24<212> DNA <213> Escherichia coli <400> 15 ccctggcgtt acccaactta atcg 24<210> 16 <211> 27 <212> DNA <213> Escherichia coli <400> 16caacatacga gccggaagca taaagtg 27 <210> 17 <211> 43 <212> DNA <213>Aspergillus oryzae <400> 17cgcaccacct tcaaaatgtc gttgaatacc gacgattccg gtc 43 <210> 18 <211> 27<212> DNA <213> Aspergillus oryzae <400> 18acctaggttc cagctaaacc cgtacac 27 <210> 19 <211> 40 <212> DNA <213>Aspergillus oryzae <400> 19 atcctgttac gcttctcaaa acgaaatctc ctccccagcc40 <210> 20 <211> 31 <212> DNA <213> Aspergillus oryzae <400> 20agctggaacc taggtgcact ccccgcgcca c 31 <210> 21 <211> 31 <212> DNA <213>Aspergillus oryzae <400> 21 cagtgagcgc aacgcaatta atgtgagtta g 31 <210>22 <211> 27 <212> DNA <213> Aspergillus oryzae <400> 22gggatgtgct gcaaggcgat taagttg 27 <210> 23 <211> 40 <212> DNA <213>Aspergillus oryzae <400> 23 ccgctgctag gcgcgccgtg cactataggg cgaattgggc40 <210> 24 <211> 44 <212> DNA <213> Aspergillus oryzae <400> 24tggggtact acaggacgta acattttgac gagctgcgga attg 44 <210> 25 <211> 43<212> DNA <213> Aspergillus oryzae <400> 25cacggcgcgc ctagcagcgg gtagtggtgg atacgtactc ctt 43 <210> 26 <211> 26<212> DNA <213> Aspergillus oryzae <400> 26 ttcaggtcac gttctaagct tatcag26 <210> 27 <211> 37 <212> DNA <213> Aspergillus oryzae <400> 27cccccctccg gatgatgtag aagttgctcg gtagctg 37 <210> 28 <211> 36 <212> DNA<213> Aspergillus oryzae <400> 28cccccctccg gacaattgcc gcgaaaaatt aaattg 36 <210> 29 <211> 24 <212> DNA<213> Aspergillus oryzae <400> 29 ccagaggtga ctttatccaa gatt 24 <210> 30<211> 44 <212> DNA <213> Aspergillus oryzae <400> 30caattccgca gctcgtcaaa atgttacgtc ctgtagaaac ccca 44 <210> 31 <211> 39<212> DNA <213> Aspergillus oryzae <400> 31tcgagctcgg tacccggtta ctgctctccc ttgatgatg 39 <210> 32 <211> 27 <212>DNA <213> Aspergillus oryzae <400> 32 taggtagtga acctatttcg agagcag 27<210> 33 <211> 45 <212> DNA <213> Aspergillus oryzae <400> 33taggttcact acctagcggc cgcacaggca ccttgcatca tcatc 45 <210> 34 <211> 37<212> DNA <213> Aspergillus oryzae <400> 34ctctagagga tccccggacc gacgattcgt tgaagag 37 <210> 35 <211> 37 <212> DNA<213> Aspergillus oryzae <400> 35aggtatcgaa ttcccgacga gctcgtacag atcatg 37 <210> 36 <211> 42 <212> DNA<213> Aspergillus oryzae <400> 36ccatgggaaa tgcccgggag agcagagtgc atggaatact ag 42 <210> 37 <211> 48<212> DNA <213> Aspergillus oryzae <400> 37gcactctgct ctcccggtgg tgggaaatct tgtatataat tgtgattg 48 <210> 38 <211>44 <212> DNA <213> Aspergillus oryzae <400> 38ccatgggaaa tgcccgggcg acactggaag aactgcttga agag 44 <210> 39 <211> 44<212> DNA <213> Aspergillus oryzae <400> 39cctgccgcga gatctgggca tacccatgg gcctaaccca aatc 44 <210> 40 <211> 45<212> DNA <213> Aspergillus oryzae <400> 40ccatgggaaa tgcccagatc tcgcggcagg gttgacacag ttgac 45 <210> 41 <211> 40<212> DNA <213> Aspergillus oryzae <400> 41gcactctgct ctcccagtaa cccattcccg gttctctagc 40 <210> 42 <211> 44 <212>DNA <213> Aspergillus oryzae <400> 42 atgtcgttga ataccgacga ttccggtc 28<210> 43 <211> 44 <212> DNA <213> Aspergillus oryzae <400> 43ggatatcgga tccccccaga ggtgacttta tccaagattc cttc 44 <210> 44 <211> 32<212> DNA <213> Aspergillus oryzae <400> 44caattgccgc gaaaaattaa attgaatcta tg 32 <210> 45 <211> 28 <212> DNA <213>Aspergillus oryzae <400> 45 gtagtggtgg atacgtactc cattatg 28 <210> 46<211> 44 <212> DNA <213> Aspergillus oryzae <400> 46tcgagctcgg tacccttcag gtcacgttct aagcttatca gctg 44 <210> 47 <211> 40<212> DNA <213> Aspergillus oryzae <400> 47cgtatccacc actaccacta tagggcgaat tgggcccgac 40 <210> 48 <211> 40 <212>DNA <213> Aspergillus oryzae <400> 48gttcaaggca gacanttga cgagctgcgg aattggtcag 40 <210> 49 <211> 40 <212><213> Aspergillus oryzae <400> 49ccaccactac cccgggaagc gtaacaggat agcctagacc 40 <210> 50 <211> 42 <212>DNA <213> Aspergillus oryzae <400> 50ctgcaggatg attagagtaa cccattcccg gttctctagc tg 42 <210> 51 <211> 33<212> DNA <213> Acremonium cellulolyticus <400> 51atgtctgcct tgaactcnt caatatgtac aag 33 <210> 52 <211> 46 <212> <213>Acremonium cellulolyticus <400> 52atcctgttac gcttcctaca aacattgaga gtagtaaggg ttcacg 46 <210> 53 <211> 26<212> DNA <213> Aspergillus oryzae <400> 53 ctaatcatcc tgcagctccg tcattg26 <210> 54 <211> 42 <212> DNA <213> Aspergillus oryzae <400> 54tatcgcggc aattggcaag ctcgagcatc caactaaact ag 42

The invention claimed is:
 1. A combination of a base sequence forprotein expression comprising: a gene encoding protein; a promoter ofthe gene, the promoter being linked upstream of the gene; and a ciselement whose activity is improved by an artificial transcriptionfactor, the cis element being linked upstream of the promoter, and abase sequence for artificial transcription factor expression comprising:a gene encoding the artificial transcription factor; and a promoter ofthe gene, the promoter being linked upstream of the gene, wherein thecis element is represented by SEQ ID NO: 1, and wherein the artificialtranscription factor comprises a DNA binding domain comprising apolynucleotide sequence of upstream 1 to 118 aa of a transcriptionfactor KojR and an active domain comprising a base polynucleotidesequence of downstream 150 to 604 aa of a transcription factor AmyR, andthe active domain is linked downstream of the DNA binding domain, and isrepresented by SEQ ID NO:
 2. 2. The base sequence for protein expressionaccording to claim 1, wherein the cis element(s) is (are) linkedupstream of the promoter of the gene encoding the protein at any numberin a range of 1 copy to 10 copies.
 3. An expression vector including acombination of a base sequence for protein expression, the base sequencefor protein expression comprising: a gene encoding protein; a promoterof the gene, the promoter being linked upstream of the gene; and a ciselement whose activity is improved by an artificial transcriptionfactor, the cis element being linked upstream of the promoter, and abase sequence for artificial transcription factor expression comprising:a gene encoding the artificial transcription factor; and a promoter ofthe gene, the promoter being linked upstream of the gene, wherein thecis element is represented by SEQ ID NO: 1, and wherein the artificialtranscription factor comprises a DNA binding domain comprising apolynucleotide sequence of upstream 1 to 118 aa of a transcriptionfactor KojR and an active domain comprising a polynucleotide sequence ofdownstream 150 to 604 aa of a transcription factor AmyR, and the activedomain is linked downstream of the DNA binding domain, and isrepresented by SEQ ID NO:
 2. 4. The expression vector according to claim3, wherein in the base sequence for protein expression, the ciselement(s) is (are) linked upstream of the promoter of the gene encodingthe protein at any number in a range of 1 copy to 10 copies.
 5. Atransformant including a combination of a base sequence for proteinexpression, the base sequence for protein expression comprising: a geneencoding protein; a promoter of the gene, the promoter being linkedupstream of the gene; and a cis element whose activity is improved by anartificial transcription factor, the cis element being linked upstreamof the promoter, and a base sequence for artificial transcription factorexpression comprising: a gene encoding the artificial transcriptionfactor; and a promoter of the gene, the promoter being linked upstreamof the gene, wherein the cis element is represented by SEQ ID NO: 1, andwherein the artificial transcription factor comprises a DNA bindingdomain comprising a polynucleotide of upstream 1 to 118 aa of atranscription factor KojR and an active domain comprising apolynucleotide sequence of downstream 150 to 604 aa of a transcriptionfactor AmyR, and the active domain is linked downstream of the DNAbinding domain, and is represented by SEQ ID NO:
 2. 6. The transformantaccording to claim 5, wherein in the base sequence for proteinexpression, the cis element(s) is (are) linked upstream of the promoterof the gene encoding the protein at any number in a range of 1 copy to10 copies.
 7. The transformant according to claim 5, wherein saidtransformant is derived from koji mold used as a host cell.
 8. Thetransformant according to claim 7, wherein the koji mold is anAspergillus oryzae HO2 strain (National Institute of Technology andEvaluation, Patent Microorganisms Depositary, #122, 2-5-8Kazusakamatari, Kisarazu-shi, Chiba, Japan, Deposition Date: Nov. 12,2013, Deposition No.: NITE BP-01750).
 9. The transformant according toclaim 7, wherein the koji mold is an Aspergillus oryzae HO4 strain(National Institute of Technology and Evaluation, Patent MicroorganismsDepositary, #122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan,Deposition Date: Dec. 9, 2014, Deposition No.: NITE BP-01980).
 10. Amethod for producing protein, comprising culturing a transformantincluding a combination of a base sequence for protein expression whichcomprises: a gene encoding the protein; a promoter of the gene, thepromoter being linked upstream of the gene; and a cis element whoseactivity is improved by an artificial transcription factor, the ciselement being linked upstream of the promoter, wherein the cis elementis represented by SEQ ID NO: 1, and a base sequence for artificialtranscription factor expression comprising: a gene encoding theartificial transcription factor; and a promoter of the gene, thepromoter being linked upstream of the gene, and recovering the proteinencoded by the gene overexpressed by the base sequence for proteinexpression, from a medium or inside of the transformant after theculture, wherein the artificial transcription factor comprises a DNAbinding domain comprising a polynucleotide sequence of upstream 1 to 118aa of a transcription factor KojR and an active domain comprising apolynucleotide sequence of downstream 150 to 604 aa of a transcriptionfactor AmyR, and the active domain is linked downstream of the DNAbinding domain, and is represented by SEQ ID NO:
 2. 11. The method forproducing protein according to claim 10, wherein in the base sequencefor protein expression, the cis element(s) is (are) linked upstream ofthe promoter of the gene encoding the protein at any number in a rangeof 1 copy to 10 copies.